The infection of macaques by simian immunodeficiency virus (SIV) results in many of the characteristics of HIV infection and progression to AIDS in humans, including loss of CD4+ T cells and susceptibility to opportunistic infections. In this animal model, individuals can be inoculated at a specific time, by defined routes, and with known amounts of molecularly characterized virus isolates. Frequent specimens can also be obtained, providing a good model for evaluating the role of viral and host factors in the progression to disease. The virus we have used was isolated from a pig-tailed macaque (Macaca nemestrina) that died with lymphoblastic lymphoma in 1982, and is named SIV/Mne. The original isolate, obtained on HuT 78 cells, is moderately pathogenic in three species of macaques (rhesus (M. mulatta), cynomolgus macaques (M. fascicularis), and pig-tailed macaques). When inoculated intravenously or mucosally, this isolate causes CD4+ cell depletion in approximately 8 to 26 months, depending on species of macaque. Our studies have focussed on attempts to develop an effective vaccine to prevent AIDS in macaques as well as to test antiviral compounds for their ability to prevent infection or decrease the viral load in infected primates, as a model to prevent and control HIV infection in humans. 1. Vaccinia Virus Recombinant AIDS Vaccines. Our infectious and pathogenic molecular clone of SIV/Mne has been used to construct various recombinant vaccinia viruses that express different regions of the virus. We previously reported that envelope (gp160)-based vaccines, when used in a recombinant vaccinia virus priming and subunit protein boosting regimen, protected almost all macaques from infection by the cloned homologous virus. The breadth of this immunity, however, appears to be limited, since approximately half of the immunized animals were infected after challenge by the uncloned virus SIV/Mne. In order to identify the regions of the virus needed for increased protection against a heterologous challenge, we designed a study in which various regions of SIV were systematically added, as recombinant vaccinia constructs, to the envelope-based vaccine regimen. These studies indicate that the inclusion of the transmembrane protein in the envelope-based vaccine is essential in order to obtain protection. In addition, the presence of both envelope and core antigens were found to now protect against a heterologous virus challenge, indicating that responses to core antigens contributed to the broadening of protective immunity. Our results argue for the inclusion of multiple antigenic targets in the design of recombinant vaccines against AIDS, and should be directly applicable to designing vaccines to protect humans against HIV. 2. Inactivated SIV with Functionally Intact Virion Surface Proteins as Vaccines. SIV was inactivated by incubation with aldrithiol-2 (AT-2), which covalently modifies the nucleocapsid protein (p8NC). The resulting virus is non-infectious and maintains conformationally and functionally intact virion surface proteins. Pig-tailed macaques were immunized with AT-2 inactivated CL E11S, leading to both cellular and humoral immune responses, and then challenged with CL E11S virus grown in pig-tailed macaque peripheral blood lymphocytes (PBMC). All control macaques became infected; the majority of the immunized macaques did not show measurable SIV RNA in plasma, and virus isolation and detection of viral sequences by PCR was only intermittent, compared to controls. These encouraging results suggest that immunization contained the pathogenic challenge virus. 3. Comparison in Pig-Tailed Macaques of Recombinant Vaccinia and AT-2 Inactivated SIV as Immunogens in Eliciting Protective Immunity against Pathogenic SIV/Mne. Of the recent vaccine strategies employed in the SIV/Mne model system, two seem particularly promising. One of these involves the use of vaccinia recombinants expressing the gag, pol, and env regions of SIV. As noted above, when used in a "prime and boost" regimen, these vaccines afford good protection against a heterologous SIV/Mne challenge. The conformationally authentic whole AT-2 inactivated virions also appear to be a promising antigen for the development of AIDS virus vaccines, and may be even more effective when used as the boost component in the prime-boost immunization regimens employing either live vaccinia vectors or DNA immunization. Both of these vaccine strategies have, however, been previously tested in different species of macaques which have been shown to differ in their susceptibility to the pathogenic effects of SIV/Mne. Studies are underway to compare these two approaches in the same species - pig-tailed macaques. An experiment has been designed to answer the following questions: (1) Which of these two vaccine approaches affords the best protection from an intravenous challenge by a heterologous pathogenic virus? (2) Is boosting necessary after vaccinia priming? (3) How does an AT-2 inactivated virus preparation compare to recombinant gag/pol/env proteins as boosting immunogen? (4) Does the density and functional integrity of the gp120 molecules on the surface of inactivated virus preparations affect vaccine efficacy? The comparison of these two vaccine approaches in a highly susceptible macaque species will address the questions posed above and determine the future direction of AIDS vaccine experiments. 4. Molecular Determinants of SIV/Mne Control in Infected Macaques. One of the necessary components of a vaccine experiment is the availability of well-characterized stocks of virus. We have isolated single-cell clones from the original culture of SIV/Mne that are being analyzed for sequence variation in a variable region of the envelope surface protein (gp120). This information is valuable for the selection of viral stocks that differ from the homologous SIV/Mne molecular clone and can be used to challenge vaccinated macaques. In addition, with the collaboration of Drs. Bruce Crise, Elena Chertova and Louis Henderson of the AIDS Vaccine Program at NCI/SAIC-Frederick, we are also examining the relative content of the surface protein gp120 in these clones, and constructing cloned viruses that contain either truncated or full-length envelope transmembrane proteins (gp32/41). These constructs will be used for pathogenesis studies, and to examine viral loads and rates of clearance of SIV after challenge. 5. Development of primate vaccines that protect against natural type D retorvirus infection. Infectious type D retroviruses (SRVs) produce a naturally occurring and often fatal disease originally observed among macaques housed at primate centers in the United States. Five neutralization types have been identified (SRV1 - SRV5), three of which have been molecularly cloned and sequenced (SRV-1, 2, and 3). These infectious type D retroviruses cause an often-fatal immunosuppressive disease in macaques that is manifested by progressive weight loss, persistent diarrhea, anemia, thymic and lymphoid atrophy, opportunistic infections, and unusual neoplasms. Since the experimental infection of macaques with SIV results in similar clinical outcomes, animals used in AIDS experiments must be free of these SRV retroviruses so as not to confound pathogenesis and vaccine studies. It has therefore become important to eradicate and prevent type D retrovirus infections among captive, group-housed macaques maintained at primate centers